Work during the coming year will involve continuation of our investigation of the function of E. coli DNA ligase, E. coli DNA polymerase I, T4 DNA polymerase and dUTPase in the repair and replication of DNA. Our specific goals are the following: a. Construction of a plasmid vector containing the DNA ligase gene which permits a high degree of overproduction of the enzyme. b. Development of an in vitro system capable of generating active T4 DNA polymerase from its inactive precursor. c. Purification of DNA polymerase I from E. coli polAex2 to homogeneity and detailed examination of the polymerase and exonuclease activities of the homogeneous enzyme. E. coli polAex2 is a "tighter" mutation than polAex1 and the defect in nick translation at restrictive temperatures would be expected to be correspondingly more severe. d. Examination of a variety of DNA polymerases by the method described for DNA polymerase I to determine whether processivity is an inherent and general feature of polymerase mechanism. e. Further examination of the relationship of dUTPase to the dnaS phenotype. BIBLIOGRAPHIC REFERENCES: Konrad, E.B. and Lehman, I.R., Mutants of Escherichia coli Defective in the Joining of Nascent DNA Fragments. In DNA Synthesis and its Replication, M. Goulian and P. Hanawalt, eds., p. 304 (1975). Konrad, E.B. and Lehman, I.R., Novel Mutants of Escherichia coli That Accumulate Very Small DNA Replicative Intermediates. Proc. Nat. Aca. Sci. USA 72, 2150 (1975).